Regulatory control of the resolution of DNA recombination intermediates during meiosis and mitosis.

The efficient and timely resolution of DNA recombination intermediates is essential for bipolar chromosome segregation. Here, we show that the specialized chromosome segregation patterns of meiosis and mitosis, which require the coordination of recombination with cell-cycle progression, are achieved by regulating the timing of activation of two crossover-promoting endonucleases. In yeast meiosis, Mus81-Mms4 and Yen1 are controlled by phosphorylation events that lead to their sequential activation. Mus81-Mms4 is hyperactivated by Cdc5-mediated phosphorylation in meiosis I, generating the crossovers necessary for chromosome segregation. Yen1 is also tightly regulated and is activated in meiosis II to resolve persistent Holliday junctions. In yeast and human mitotic cells, a similar regulatory network restrains these nuclease activities until mitosis, biasing the outcome of recombination toward noncrossover products while also ensuring the elimination of any persistent joint molecules. Mitotic regulation thereby facilitates chromosome segregation while limiting the potential for loss of heterozygosity and sister-chromatid exchanges.

Alternative translation initiation by ribosomal leaky scanning produces multiple isoforms of the Pif1 helicase.

In budding yeast, the integrity of both the nuclear and mitochondrial genomes relies on dual-targeted isoforms of the conserved Pif1 helicase, generated by alternative translation initiation (ATI) of PIF1 mRNA from two consecutive AUG codons flanking a mitochondrial targeting signal. Here, we demonstrate that ribosomal leaky scanning is the specific ATI mechanism that produces not only these, but also novel, previously uncharacterized Pif1 isoforms. Both in-frame, downstream AUGs as well as near-cognate start codons contribute to the generation of these alternative isoforms. This has crucial implications for the rational design of genuine separation-of-function alleles and provides an explanation for the suboptimal behaviour of the widely employed mitochondrial- (pif1-m1) and nuclear-deficient (pif1-m2) alleles, with mutations in the first or second AUG codon, respectively. We have taken advantage of this refined model to develop improved versions of these alleles, which will serve as valuable tools to elucidate novel functions of this helicase and to disambiguate previously described genetic interactions of PIF1 in the context of nuclear and mitochondrial genome stability.

Concurrent D-loop cleavage by Mus81 and Yen1 yields half-crossover precursors.

Homologous recombination involves the formation of branched DNA molecules that may interfere with chromosome segregation. To resolve these persistent joint molecules, cells rely on the activation of structure-selective endonucleases (SSEs) during the late stages of the cell cycle. However, the premature activation of SSEs compromises genome integrity, due to untimely processing of replication and/or recombination intermediates. Here, we used a biochemical approach to show that the budding yeast SSEs Mus81 and Yen1 possess the ability to cleave the central recombination intermediate known as the displacement loop or D-loop. Moreover, we demonstrate that, consistently with previous genetic data, the simultaneous action of Mus81 and Yen1, followed by ligation, is sufficient to recreate the formation of a half-crossover precursor in vitro. Our results provide not only mechanistic explanation for the formation of a half-crossover, but also highlight the critical importance for precise regulation of these SSEs to prevent chromosomal rearrangements.

SUMOylation modulates eIF5A activities in both yeast and pancreatic ductal adenocarcinoma cells.

BACKGROUND: The eukaryotic translation initiation protein eIF5A is a highly conserved and essential factor that plays a critical role in different physiological and pathological processes including stress response and cancer. Different proteomic studies suggest that eIF5A may be a small ubiquitin-like modifier (SUMO) substrate, but whether eIF5A is indeed SUMOylated and how relevant is this modification for eIF5A activities are still unknown. METHODS: SUMOylation was evaluated using in vitro SUMOylation assays, Histidine-tagged proteins purification from His6-SUMO2 transfected cells, and isolation of endogenously SUMOylated proteins using SUMO-binding entities (SUBES). Mutants were engineered by site-directed mutagenesis. Protein stability was measured by a cycloheximide chase assay. Protein localization was determined using immunofluorescence and cellular fractionation assays. The ability of eIF5A1 constructs to complement the growth of Saccharomyces cerevisiae strains harboring thermosensitive mutants of a yeast EIF5A homolog gene (HYP2) was analyzed. The polysome profile and the formation of stress granules in cells expressing Pab1-GFP (a stress granule marker) by immunofluorescence were determined in yeast cells subjected to heat shock. Cell growth and migration of pancreatic ductal adenocarcinoma PANC-1 cells overexpressing different eIF5A1 constructs were evaluated using crystal violet staining and transwell inserts, respectively. Statistical analysis was performed with GraphPad Software, using unpaired Student's t-test, or one-way or two-way analysis of variance (ANOVA). RESULTS: We found that eIF5A is modified by SUMO2 in vitro, in transfected cells and under endogenous conditions, revealing its physiological relevance. We identified several SUMO sites in eIF5A and found that SUMOylation modulates both the stability and the localization of eIF5A in mammalian cells. Interestingly, the SUMOylation of eIF5A responds to specific stresses, indicating that it is a regulated process. SUMOylation of eIF5A is conserved in yeast, the eIF5A SUMOylation mutants are unable to completely suppress the defects of HYP2 mutants, and SUMOylation of eIF5A is important for both stress granules formation and disassembly of polysomes induced by heat-shock. Moreover, mutation of the SUMOylation sites in eIF5A abolishes its promigratory and proproliferative activities in PANC-1 cells. CONCLUSIONS: SUMO2 conjugation to eIF5A is a stress-induced response implicated in the adaptation of yeast cells to heat-shock stress and required to promote the growth and migration of pancreatic ductal adenocarcinoma cells.

Canonical and novel non-canonical activities of the Holliday junction resolvase Yen1.

Yen1 and GEN1 are members of the Rad2/XPG family of nucleases that were identified as the first canonical nuclear Holliday junction (HJ) resolvases in budding yeast and humans due to their ability to introduce two symmetric, coordinated incisions on opposite strands of the HJ, yielding nicked DNA products that could be readily ligated. While GEN1 has been extensively characterized in vitro, much less is known about the biochemistry of Yen1. Here, we have performed the first in-depth characterization of purified Yen1. We confirmed that Yen1 resembles GEN1 in many aspects, including range of substrates targeted, position of most incisions they produce or the increase in the first incision rate by assembly of a dimer on a HJ, despite minor differences. However, we demonstrate that Yen1 is endowed with additional nuclease activities, like a nick-specific 5'-3' exonuclease or HJ arm-chopping that could apparently blur its classification as a canonical HJ resolvase. Despite this, we show that Yen1 fulfils the requirements of a canonical HJ resolvase and hypothesize that its wider array of nuclease activities might contribute to its function in the removal of persistent recombination or replication intermediates.

Detection of SARS-CoV-2 in a dog with hemorrhagic diarrhea.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has infected several animal species, including dogs, presumably via human-to-animal transmission. Most infected dogs reported were asymptomatic, with low viral loads. However, in this case we detected SARS-CoV-2 in a dog from the North African coastal Spanish city of Ceuta presenting hemorrhagic diarrhea, a disease also reported earlier on in an infected dog from the USA. CASE PRESENTATION: In early January 2021, a West Highland Terrier pet dog from Ceuta (Spain) presented hemorrhagic diarrhea with negative tests for candidate microbial pathogens. Since the animal was in a household whose members suffered SARS-CoV-2 in December 2020, dog feces were analyzed for SARS-CoV-2, proving positive in a two-tube RT-PCR test, with confirmation by sequencing a 399-nucleotide region of the spike (S) gene. Furthermore, next-generation sequencing (NGS) covered > 90% SARS-CoV-2 genome sequence, allowing to classify it as variant B.1.177. Remarkably, the sequence revealed the Ile402Val substitution in the spike protein (S), of potential concern because it mapped in the receptor binding domain (RBD) that mediates virus interaction with the cell. NGS reads mapping to bacterial genomes showed that the dog fecal microbiome fitted best the characteristic microbiome of dog's acute hemorrhagic diarrhea. CONCLUSION: Our findings exemplify dog infection stemming from the human SARS-CoV-2 pandemic, providing nearly complete-genome sequencing of the virus, which is recognized as belonging to the B.1.177 variant, adding knowledge on variant circulation in a geographic region and period for which there was little viral variant characterization. A single amino acid substitution found in the S protein that could have been of concern is excluded to belong to this category given its rarity and intrinsic nature. The dog's pathology suggests that SARS-CoV-2 could affect the gastrointestinal tract of the dog.

Dual control of Yen1 nuclease activity and cellular localization by Cdk and Cdc14 prevents genome instability.

The careful orchestration of cellular events such as DNA replication, repair, and segregation is essential for equal distribution of the duplicated genome into two daughter cells. To ensure that persistent recombination intermediates are resolved prior to cell division, the Yen1 Holliday junction resolvase is activated at anaphase. Here, we show that the master cell-cycle regulators, cyclin-dependent kinase (Cdk) and Cdc14 phosphatase, control the actions of Yen1. During S phase, Cdk-mediated phosphorylation of Yen1 promotes its nuclear exclusion and inhibits catalytic activity by reducing the efficiency of DNA binding. Later in the cell cycle, at anaphase, Cdc14 drives Yen1 dephosphorylation, leading to its nuclear relocalization and enzymatic activation. Using a constitutively activated form of Yen1, we show that uncontrolled Yen1 activity is detrimental to the cell: spatial and temporal restriction of Yen1 protects against genotoxic stress and, by avoiding competition with the noncrossover-promoting repair pathways, prevents loss of heterozygosity.

Exo1 phosphorylation inhibits exonuclease activity and prevents fork collapse in rad53 mutants independently of the 14-3-3 proteins.

The S phase checkpoint is crucial to maintain genome stability under conditions that threaten DNA replication. One of its critical functions is to prevent Exo1-dependent fork degradation, and Exo1 is phosphorylated in response to different genotoxic agents. Exo1 seemed to be regulated by several post-translational modifications in the presence of replicative stress, but the specific contribution of checkpoint-dependent phosphorylation to Exo1 control and fork stability is not clear. We show here that Exo1 phosphorylation is Dun1-independent and Rad53-dependent in response to DNA damage or dNTP depletion, and in both situations Exo1 is similarly phosphorylated at multiple sites. To investigate the correlation between Exo1 phosphorylation and fork stability, we have generated phospho-mimic exo1 alleles that rescue fork collapse in rad53 mutants as efficiently as exo1-nuclease dead mutants or the absence of Exo1, arguing that Rad53-dependent phosphorylation is the mayor requirement to preserve fork stability. We have also shown that this rescue is Bmh1-2 independent, arguing that the 14-3-3 proteins are dispensable for fork stabilization, at least when Exo1 is downregulated. Importantly, our results indicated that phosphorylation specifically inhibits the 5' to 3'exo-nuclease activity, suggesting that this activity of Exo1 and not the flap-endonuclease, is the enzymatic activity responsible of the collapse of stalled replication forks in checkpoint mutants.

A comparative analysis of the intrauterine transcriptome in fertile and subfertile mares using cytobrush sampling.

BACKGROUND: Subfertility is a major problem in modern horse breeding. Especially, mares without clinical signs of reproductive diseases, without known uterine pathogens and no evidence of inflammation but not becoming pregnant after several breeding attempts are challenging for veterinarians. To obtain new insights into the cause of these fertility problems and aiming at improving diagnosis of subfertile mares, a comparative analysis of the intrauterine transcriptome in subfertile and fertile mares was performed. Uterine cytobrush samples were collected during estrus from 57 mares without clinical signs of uterine diseases. RNA was extracted from the cytobrush samples and samples from 11 selected subfertile and 11 fertile mares were used for Illumina RNA-sequencing. RESULTS: The cytobrush sampling was a suitable technique to isolate enough RNA of high quality for transcriptome analysis. Comparing subfertile and fertile mares, 114 differentially expressed genes (FDR = 10%) were identified. Metascape enrichment analysis revealed that genes with lower mRNA levels in subfertile mares were related to 'extracellular matrix (ECM)', 'ECM-receptor interaction', 'focal adhesion', 'immune response' and 'cytosolic calcium ion concentration', while DEGs with higher levels in subfertile mares were enriched for 'monocarboxyl acid transmembrane transport activity' and 'protein targeting'. CONCLUSION: Our study revealed significant differences in the uterine transcriptome between fertile and subfertile mares and provides leads for potential uterine molecular biomarkers of subfertility in the mare.

Dbf4-dependent kinase and the Rtt107 scaffold promote Mus81-Mms4 resolvase activation during mitosis.

DNA repair by homologous recombination is under stringent cell cycle control. This includes the last step of the reaction, disentanglement of DNA joint molecules (JMs). Previous work has established that JM resolving nucleases are activated specifically at the onset of mitosis. In case of budding yeast Mus81-Mms4, this cell cycle stage-specific activation is known to depend on phosphorylation by CDK and Cdc5 kinases. Here, we show that a third cell cycle kinase, Cdc7-Dbf4 (DDK), targets Mus81-Mms4 in conjunction with Cdc5-both kinases bind to as well as phosphorylate Mus81-Mms4 in an interdependent manner. Moreover, DDK-mediated phosphorylation of Mms4 is strictly required for Mus81 activation in mitosis, establishing DDK as a novel regulator of homologous recombination. The scaffold protein Rtt107, which binds the Mus81-Mms4 complex, interacts with Cdc7 and thereby targets DDK and Cdc5 to the complex enabling full Mus81 activation. Therefore, Mus81 activation in mitosis involves at least three cell cycle kinases, CDK, Cdc5 and DDK Furthermore, tethering of the kinases in a stable complex with Mus81 is critical for efficient JM resolution.

Validation of a Questionnaire to Analyze the Expectations of First-Year Nursing Students.

AIM: The aim of this study was to design and validate a questionnaire to analyze students' expectations, along with their level of interest and overall satisfaction with their nursing studies. BACKGROUND: The expectations of students on entering university are closely related to the level of interest that they have in their area of study and their general satisfaction with it. This is certainly a conditioning factor in their learning. In this regard, there are few quantitative instruments that measure the expectations of first-year nursing students. METHOD: A mixed questionnaire was constructed using items validated in previous studies, along with new items formulated following a review of the literature. The questionnaire was validated with a sample of 339 first-year degree program nursing students. RESULTS: The scales of expectations and degree of interest showed strong internal consistency (Cronbach's α > .7). CONCLUSION: The questionnaire to evaluate the expectations and interest of students demonstrated satisfactory psychometric properties.

Violence in Honduras from 2008 to 2018.

The current study documents homicide trends in Honduras from 2008 to 2018. Specifically, this study describes demographics of homicide victims and incident profiles (ie, weapons) using homicide data from the Honduras National Police and census data from the National Institute of Statistics. A total of 58 543 homicide incidents were analysed. Results indicated that the homicide rate in Honduras increased from 2008 to 2011 and decreased substantially after 2011. In addition, the male homicide victimisation rate was significantly higher than the female homicide victimisation rate across the entire time period, with the highest rate for males aged 30-44 (233.4 per 100 000 population). Firearms were the weapons used most frequently in homicides (80.3%). Implications of the findings are discussed in light of public safety in Honduras.

Positive Mental Health and Self-Care in Patients with Chronic Physical Health Problems: Implications for Evidence-based Practice.

BACKGROUND: The capacity for self-care and positive mental health (PMH) has an influence on well-being and on one's approach to chronic illness. PURPOSE: The aim was to determine the level of PMH and self-care agency as well as the relations among sociodemographic variables, PMH, and the level of self-care among patients with chronic physical health problems. We also examined correlations between PMH and self-care agency. METHODS: A descriptive, cross-sectional correlational study was conducted with a sample of 209 patients at a primary care center. The instruments used were the Positive Mental Health Questionnaire and the Appraisal of Self-Care Agency scale. The STROBE statement was used. RESULTS: Significant differences were found in the PMH factors in relation to sociodemographic variables and health conditions. Suffering one or more chronic diseases was associated negatively, and significantly, with the capacity for self-care. The four most prevalent chronic health problems in the sample were hypertension, hypercholesterolemia, obesity, and diabetes mellitus. LINKING EVIDENCE TO ACTION: In people with chronic physical health problems, there is a positive relationship between PMH and self-care capacity. An increase in the possibility of caring for oneself saw an increase in PMH; conversely, an increase in PMH brought with it an increased capacity for self-care as well. Therefore, if actions are taken to increase PMH, the capacity for self-care will also increase.

Study of the effect of anti-rhGAA antibodies at low and intermediate titers in late onset Pompe patients treated with ERT.

Late onset Pompe disease (LOPD) is a genetic disorder characterized by slowly progressive skeletal and respiratory muscle weakness. Symptomatic patients are treated with enzyme replacement therapy (ERT) with alglucosidase alpha (rhGAA). Although most of ERT treated patients develop antibodies against rhGAA, their influence on clinical progression is not completely known. We studied the impact of anti-rhGAA antibodies on clinical progression of 25 ERT treated patients. We evaluated patients at visit 0 and, after 1 year, at visit 1. We performed several muscle function tests, conventional spirometry and quantitative muscle MRI (qMRI) using 3-point Dixon analysis of thigh muscles at both visits. We also obtained serum samples at both visits and anti-rhGAA antibodies were quantified using ELISA. Antibody titers higher than 1:200 were identified in 18 patients (72%) of our cohort. Seven patients (28%) did not develop antibodies (0 to <1:200), 17 patients (68%) developed low to intermediate titers (1:200 to <1:31,200) and 1 patient (4%) developed high titers (>1:31,200). We analyzed the effect of low and intermediate antibody titers in clinical and radiological progression. There were no differences between the results of muscle function tests, spirometry or fat fraction analyzed using qMRI between patients with and without antibodies groups at baseline. Moreover, antibody titers did not influence muscle function test, spirometry results or qMRI results at year 1 visit. Most of the LOPD patients developed antibodies against ERT that persisted over time at low or intermediate levels. However, antibodies at these low and intermediate titers might not influence clinical response to the drug.

Mechanism of Holliday junction resolution by the human GEN1 protein.

Holliday junction (HJ) resolution is essential for chromosome segregation at meiosis and the repair of stalled/collapsed replication forks in mitotic cells. All organisms possess nucleases that promote HJ resolution by the introduction of symmetrically related nicks in two strands at, or close to, the junction point. GEN1, a member of the Rad2/XPG nuclease family, was isolated recently from human cells and shown to promote HJ resolution in vitro and in vivo. Here, we provide the first biochemical/structural characterization of GEN1, showing that, like the Escherichia coli HJ resolvase RuvC, it binds specifically to HJs and resolves them by a dual incision mechanism in which nicks are introduced in the pair of continuous (noncrossing) strands within the lifetime of the GEN1-HJ complex. In contrast to RuvC, but like other Rad2/XPG family members such as FEN1, GEN1 is a monomeric 5'-flap endonuclease. However, the unique feature of GEN1 that distinguishes it from other Rad2/XPG nucleases is its ability to dimerize on HJs. This functional adaptation provides the two symmetrically aligned active sites required for HJ resolution.

Association between periodontitis and ischemic stroke: a systematic review and meta-analysis.

Several observational studies have suggested an association between periodontitis and cerebral ischemia. This meta-analysis aimed to investigate whether this link exists, and if so, the degree to which it is significant. The Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guideline for systematic review was used. The search strategy included using electronic databases and hand searching works published up to March 2015. MEDLINE via PubMed, EMBASE, Proceedings Web of Science and Current Contents Connect were searched by two independent reviewers. Case-control, cross-sectional or cohort studies including patients with measures of periodontitis and ischemic stroke were eligible to be included in the analysis. Quality assessments of selected studies were performed. From a total of 414 titles and abstracts, 57 potentially relevant full text papers were identified. After inclusion criteria were applied, 8 studies were included in the present systematic review (5 case-control and 3 cohort studies). Although it was not the intention, cross-sectional studies were excluded due to eligibility criteria were not accomplished. Therefore, meta-analyses were conducted with data retrieved from the 8 studies included. These meta-analyses showed statistically significant association between periodontitis and ischemic stroke in both cohort pooled relative risks at 2.52 (1.77-3.58), and case-control studies pooled relative risks at 3.04 (1.10-8.43). In conclusion, the present meta-analysis demonstrated an association between periodontitis and ischemic stroke. However, well-designed prospective studies should be carried out to provide robust evidence of the link between both diseases. In regards to ischemic stroke subtypes, further case-control studies should be carried out to investigate whether there is any association between the different subtypes of cerebral infarcts and periodontitis.

Evaluation of mechanical closure resistance of sutureless vitrectomy sclerotomies after conjunctival cauterization with bipolar diathermy forceps.

BACKGROUND: Suturing is the most widely used technique to close leaking sclerotomies after transconjunctival sutureless vitrectomy (TSV). However, with the aim of avoiding the disadvantages caused by conjunctival stitches, there have been described other closure techniques, such as the cauterization of the conjunctiva placed over the incisions. To continue advancing knowledge of the incisional occlusion effect achieved by conjunctival diathermy, it would be also interesting to study the wound closure resistance obtained under intraocular pressure (IOP) changes, given that in the early postoperative period eyes are subjected to pressure stress. In our study, we compare the mechanical resistance observed in sclerotomies treated with bipolar diathermy after TSV compared to that found in incisions in which cauterization was not performed. METHODS: This was an experimental, randomized, and observer-masked study in which 23-gauge TSV was performed in 80 cadaveric pig eyes. Once each vitrectomy was finished, cauterization was performed with bipolar diathermy forceps on the conjunctiva placed over one of the superior sclerotomy sites; no maneuver was performed over the other superior incision. IOP was gradually increased by means of the vitrectomy system (Accurus; Alcon Laboratories, TX) until one of the superior sclerotomies opened, allowing internal ocular solution to escape. RESULTS: In 35 % of cases (28 of 80 eyes), sclerotomies subjected to diathermy allowed intraocular fluid escape first (p = 0.01). When comparing opening pressure values, cauterized incisions leaked at significantly higher pressure levels than those in which diathermy was not applied (p < 0.001). CONCLUSIONS: Bipolar diathermy on sutureless sclerotomies has demonstrated to be, in our experimental model, an effective method for increasing the sclerotomy closure resistance. Although its use in vitrectomized eyes has previously been described, our study is the first to analyze the response of cauterized sclerotomies to IOP increases.

Carotid Intima-Media Thickness is Not Associated with Markers of Atherosclerosis in Stroke Patients.

BACKGROUND: It has been argued that carotid intima-media thickness (IMT) could better reflect an adaptive response of the vessel wall rather than being a marker of atherosclerosis. We explore this hypothesis by analyzing the ARTICO data. METHODS: The ARTICO study was designed to evaluate the prognostic value of the pathological ankle-brachial index (ABI) for the emergence of new vascular events in patients who have suffered a noncardioembolic stroke. Collected variables were as follows: vascular risk factors, mean waist perimeter, quantification of carotid IMT, characteristics of carotid plaques, ABI, and presence of microalbuminuria. RESULTS: A total of 591 patients with a complete carotid evaluation were available. There was no correlation between ABI and IMT (Spearman's, p NS). Logistic regression revealed that pathological ABI correlated significantly only with internal carotid artery stenosis greater than or equal to 50% (OR [odds ratio] 2.80, 1.66-4.71, P < .01) and peripheral artery disease (OR 3.33, 1.63-6.78, P < .01). However, multivariate regression analysis demonstrated that carotid IMT was independently associated with age (OR 1.05, 95% confidence interval [CI] 1.02-1.09, P < .01), hypertension (OR 1.83, 95% CI 1.02-3.26, P = .04), waist circumference (OR 1.03, 95% CI 1.01-1.05, P < .01), and microalbuminuria (OR 2.02, 95% CI 1.22-3.35, P < .01). CONCLUSION: In our patients, carotid IMT does not seem to be associated with unequivocal markers of atheromatosis such as the existence of relevant carotid plaques or pathological ABI. These results as well as the association of IMT with age, hypertension, microalbuminuria, and mean waist perimeter support the hypothesis that IMT must be considered a risk factor for general vascular disease rather than a marker of atherosclerotic burden.

Association between macular perfusion and photoreceptor layer status in diabetic macular edema.

PURPOSE: To evaluate the relationship between the photoreceptor layer status (inner segment ellipsoid band and external limiting membrane) and the foveal avascular zone size, as a result of macular perfusion, in patients with diabetic macular edema. METHODS: This observational case series study included 151 eyes of 118 patients with naive diabetic macular edema. The length of the disrupted photoreceptor layer was assessed by optical coherence tomography. The foveal avascular zone diameter was measured on fluorescein angiogram. RESULTS: No significant association was found between the foveal avascular zone size and the mean lengths of the disrupted inner segment ellipsoid band nor the external limiting membrane in patients with naive diabetic macular edema. CONCLUSION: Macular ischemia, which lengthens the distance from the perifoveal vessels to the center of the fovea and may disrupt the normal flow of nutrients by simple diffusion to the photoreceptor line, does not seem to influence on inner segment ellipsoid band nor external limiting membrane integrity. Future studies may evaluate the effect of choroidal vascularization on the photoreceptor layer status to enhance the knowledge about the photoreceptor layer nutrients source.

Molecular epidemiology and virulence of Escherichia coli O16:H5-ST131: comparison with H30 and H30-Rx subclones of O25b:H4-ST131.

The present study was carried out to evaluate the prevalence of the clonal subgroup O16:H5-ST131 and the H30 and H30-Rx subclones among E. coli isolates causing extraintestinal infections and to know their virulence potential. The ST131 clonal group accounted for 490 (16%) of the 2995 isolates obtained from clinical samples in five Spanish hospitals during the study period (2005-2012). Among those 490 ST131 isolates, 456 belonged to serotype O25b:H4, 27 to O16:H5 and seven were O-non-typeable:H4 (ONT:H4). All 27 O16:H5 isolates showed fimH41, whereas fimH30 and fimH22 alleles were the most frequently detected among O25b:H4 isolates. The majority (381/490; 78%) of ST131 isolates belonged to H30 subclone, and 302 of 381 (79%) H30 isolates belonged to the H30-Rx subclone. Of the 27 O16:H5 isolates, 48% produced CTX-M-14; however, none produced CTX-M-15. In contrast, 46% of O25b:H4 isolates produced CTX-M-15 while only 2% produced CTX-M-14. More than a half of the O16:H5 isolates (56%) showed the ExPEC status which was significantly more prevalent within O25b:H4 isolates (81%) (P<0.01), especially among H30-Rx (97%) isolates. In the present study, a modified virotype scheme was applied within which approximately half (52%) of the O16:H5 isolates showed the C1 specific virotype. Despite their low virulence-gene score (mean of virulence genes 6.4 versus 8.5 in O25b:H4 isolates), six out of the 10 O16:H5 isolates assayed showed high virulence in the mouse model of sepsis (killed 90-100% of mice challenged). Furthermore, four O16:H5 isolates of virotypes A and C1, carrying K2 variant of group II capsule, showed lethality at 24h. Thus, certain O16:H5 fimH41 isolates show a similar in vivo virulence to that reported with the highly virulent O25b:H4 H30-Rx isolates (Mora et al., PLOS ONE 2014, e87025), supporting their potential virulence for humans.